The present invention relates to techniques for preserving a biological cell specimen.
The preservation of viable cell specimens is a very important field of science. It is essential to long-term preservation of tissue specimens, sperm, and fertilized ovum (zygotes), among others. Cooling of cell specimens to cryogenic temperatures, and then holding the specimens at cryogenic temperatures is typically used for preservation. The cooling must be carefully controlled to minimize crystal size so that crystals are prevented from attaining sizes which are capable of damaging cells. During cooling, the temperature range of from room temperature to about -40 to -50 degrees Celsius is critical for crystal formation. Precise control of cooling in this range is essential to the viability of the specimen. The most critical temperature range is from about zero degrees C to a few degrees below -8.degree. C. In this range, a specimen releases heat of fusion during cooling. Thus, in this range, the amount of heat which must be removed to maintain a programmed cooling gradient increases markedly. As a consequence, the rate of heat removal requires precise control to maintain a desired cooling rate through this range. Once the speciment is cooled below about -40.degree. or -50.degree. C., crystal formation is no longer a problem, and thus less precise control of the cooling rate is permitted.
The cooling parameters for different lines of cells may differ. Thus, the cooling programs (temperatures, rates and times) for different cell lines may also differ.
At the present, two ways to achieve the cooling of the cells are employed: cooling with a liquified gas (typically liquid nitrogen) and cooling by mechanical refrigeration.
When liquid nitrogen is employed, it is possible to achieve a very high temperature gradient. For example, an initial cooling rate of as much as 80.degree. C. per minute is possible. The specimen is usually dipped in a container with liquid nitrogen. Although this gives a large cooling rate, the cooling rate cannot be controlled. Also, liquid nitrogen presents a problem in handling.
Better control of cooling is provided using vapor from a liquified gas (liquid nitrogen). The specimen to be cooled is placed in a vessel with the liquid nitrogen, but out of physical contact with the liquid. The cold vapor coming from the liquid nitrogen is relied on for cooling. This method suffers from the low thermal capacity of a gas. As a consequence, it is difficult to maintain uniform cooling rates and temperatures across a specimen.
Mechanical cooling employs a refrigeration unit that uses coolant coils about the walls of a specimen container. A heat transfer medium, such as, for example, ethanol, in the specimen container aids in the transfer of heat from the specimen to the coolant coils. This technique does not need liquid nitrogen, and can provide cooling down to at least -80.degree. C. However, the temperature gradient attainable by mechanical refrigeration is limited to about 2-3 degrees Celsius per minute. For many biological specimens, this rate is not great enough to prevent the formation of crystals of damaging size through the critical temperature range above -40.degree. C.